Effect of phentolamine and preperfusion with glucose on insulin release from the isolated perfused pancreas from fasted and fed rats

Diabetologia, Oct 1975

Starvation of rats for 24 hrs resulted in decreased insulin release from the isolated rat pancreas. The effect of fasting could not be counteracted by elevation of the glucose level in the equilibration medium from 0.8 to 1.5 mg/ml. The alpha-adrenergic blocking agent phentolamine (10 μg/ml) stimulated glucose induced insulin release to approximately the same extent in fasted as in fed rats. These findings illustrate the importance of endogenous catecholamines in the regulation of insulin secretion from the isolated pancreas. Our experiments suggest that the impairment of insulin secretion on fasting is due neither to the inhibitory effect of catecholamines nor to the lack of substrate in the pancreas at the initiation of the stimulation.

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Effect of phentolamine and preperfusion with glucose on insulin release from the isolated perfused pancreas from fasted and fed rats

Effect of P h e n t o l a m i n e and Preperfusion with Glucose on Insulin R e l e a s e from the Isolated Perfused Pancreas from Fasted and F e d Rats S. Efendi 0 E. Cerasi 0 R. Luft 0 0 Dept. of Endocrinology and Metabolism, Karolinska Hospital , Stockholm , Sweden Summary. Starvation of rats for 24 hrs resulted in decreased insulin release from the isolated rat pancreas. The effect of fasting could not be counteracted by elevation of the glucose level in the equilibration medium from 0.8 to 1.5 mg/ml. The alpha-adrenergic blocking agent phentolamine (10 vtg/ml) stimulated glucose induced insulin release to approximately the same extent in fasted as in fed rats. These findings illustrate the importance of endogenous catecholamines in the regulation of in- Insulin release; perfused pancreas; fasting; phentolamine - The modulating effect of the adrenergic nervous system and circulating catecholamines on insulin secretion has been amply demonstrated [ 1-5 ]. Isolated preparations of pancreatic tissue for the study of insulin release have been used with the assumption that such an effect is negligible in vitro. However, recent studies have established that considerable quantities of catecholamines may be liberated from the isolated peffused dog pancreas [ 6 ]. The present study was undertaken in order to evaluate the role of endogenous pancreatic catecholamines in glucose induced insulin output from the isolated rat pancreas. Furthermore, since data in the literature suggest that the diminished insulin responsiveness of the fasted animals may be due to an increased action of pancreatic monoamines [ 7 ], the study was extended to fasted rats. Materials and Methods Sprague-Dawley rats, weighing 200-250 g, fed ~ t libitum or fasted for 24 hrs, were used for the preparation of the perfused isolated pancreas. The animals were anaesthetized by intraperitoneal injection of 50 mg/kg of pentobarbital, and the pancreas was isolated by a slight modification of the technique of Loubati6res et al. [ 8 ]. The pancreas was completely separated from adjacent organs and all vessels were carefully ligated. The gland was perfused with a Krebs-Ringer bicarbonate solution, containing 0.8 or 1.5 g/1 of glucose and 20 g/1 of beef albumin (fraction sulin secretion from the isolated pancreas. Our experiments suggest that the impairment of insulin secretion on fasting is due neither to the inhibitory effect of catecholamines nor to the lack of substrate in the pancreas at the initiation of the stimulation. V, Armour Co). The final solution was adjusted to pH 7.4 with 0.1 N HCI, and then continuously gassed with a mixture of 95% oxygen and 5% carbon dioxide. The perfusate was administered into the coeliac artery and run into the prepared pancreas by an open circuit, non-recycling, perfusion system. The portal effluent was collected every 60 sec. Flow rates were kept approximately at 2.5 ml/min by making minor changes in arterial pressure. A 40 min equilibration period preceded the stimulatory concentration of glucose. Phentolamine (Regitine| Ciba) and adrenaline (Astra AB, S6dert~ilje, Sweden) were added to the perfusate as indicated in the figures. Insulin was determined by a double-antibody radioimmunoassay using insulin reagent kits (Radiochemical Centre, Amersham) and a rat insulin standard, kindly provided by Dr. J. Schlichtkrull (Novo Research Institute, Copenhagen). The effect of phentolamine on insulin release was evaluated by comparison of the areas under the insulin curves. The initial phase of insulin corresponds to 0 to 7 min after the start of the infusion, while the second phase comprises the period beweeen 7 and 17 min. Results As seen in Fig. 1, increasing the glucose concentration in the perfusate from 1.5 mg to 3.0 mg/ml was accompanied by a prominent and rapid insulin release. The peak insulin value was at 2 min, with a nadir at about 4 min, after which the insulin secretion rate insulin /zUlml 2000 1500 (n=7) (n=3) --.,.., -20-10 0 10 20 30 -20-10 0 10 20 30 -20-10 0 10 20 30 [glucose [ [ glucose I I gtuc~ [ [ adrenaline [ r a d r e n a l i n e [phentoiamine 3500 continuously increased during glucose infusion. The !i/~ In:5) addition of adrenaline at a concentration of 4 ng/ml markedly suppressed the glucose induced insulin release. This inhibition was counteracted by the alpha3000 adrenergic blocking agent, phentolamine (10 ~tg/ml), )' which was added to the perfusate 5 rain after the application of adrenaline. 2500 The effect of the above dose of phentolamihe on 1,11oioo11Glu1coseexerted a much more pronounced insulin I glucose induced insulin release, in the absence of pinAsJu/lminin adrenaline, is demonstrated in Fig. 2, the left hand panel of which summarizes the experiments on fasted rats, while the right o n e gives the results obtained in F rats fed ad libitum. In both types of experiments the equilibration medium contained 0.8 mg/ml of glucose. release in the fed rats where the release rates both at In=5} the initial and second phase were more than three times those obtained in the fasted animals (p<0.005) (Table 1). Phentolamine significantly increased the insulinogenic effect of glucose independently of the nutritional state of the rats. This enhancement of the effect of glucose was about 100 per cent for the early ~ n 6) Increasing the glucose concentration in the equilias well as the late phase of insulin release (Table 1). bration medium from 0.8 to 1.5 mg/ml in fasted rats almost doubled both the early and late phase of insulin i 9 release induced by 3.0 mg/ml of glucose (Fig. 3, Table -10 0 10 20 -10 0 10 20 1). Phentolamine exerted a quantitatively identical [phentolomine IPhentolemine] enhancing effect on glucose induced insulin release g ucose Igluc~ I under these experimental conditions. Insulin release after 40 min of equilibration was significantly higher in fed than in fasted rats (p< 0.01) (Table 1). Phentolamine significantly enhanced basal insulin release in both fed rats and fasted rats when the equilibration medium contained 1.5 mg/ml of glucose (Table 1). 0,J & Glucose conc. in mg/ml during Insulin release ~tU/ml equit, stim. Phentolamine basal a 3.0 3.0 3.0 3.0 3.0 3.0 + + + son and Porte [ 5 ] showed that administration of alphaadrenergic blockers stimulated, while beta-adrenergic blockers inhibited basal insulin secretion in man. We have previously demonstrated that the human pancreas is sensitive to small amounts of adrenaline [ 4 ]. The above experiments were performed in vivo, which makes it difficult to estimate the relative efficiency of catecholamines liberated at the nerve endings of the islets and catecholamines reaching the pancreas via the circulation. The present findings, that phentoiamine enhanced glucose induced insulin secretion by approximately 100% in the isolated rat pancreas suggest that even tissue-bound pancreatic catecholamines may be of major significance in the regulation of insulin release. Although this statement is supported by the demonstration that the isolated pancreas may contain large quantities of catecholamines [ 6 ], it cannot be excluded that the observed action of phentolamine on insulin release is a direct one on the B-cell, i.e., not mediated by the blockade of the suppression exerted by endogenous catecholamines. In accordance with previous observations [ 4, 9-14 ], the present studies show that short-term fasting (24 h) was accompanied by decreased glucose induced insulin release. The kinetics of insulin release were not modified. This has also been demonstrated by Fink et al. [ 14 ]. It has been shown that the impaired insulin release during fasting can be partially corrected by reserpine, and the possible role of pancreatic monoamines in the mediation of this derangement has been discussed [ 7 ]. In the present studies the defective insulin release a Insulin release after preincubation for 40 min. b Integrated insulin response, in ~tU X min X m1-1 o 1; 2; phentotamine gucose It has been well established, in vivo as well as in vitro, that adrenaline is a potent inhibitor of insulin release via its alpha-adrenergic effect on the pancreatic beta-cell [ 1-5 ]. Recent reports suggest that catecholamines might also regulate insulin release under physiological conditions [ 4, 5 ]. Thus, Robertafter fasting could not be normalized by addition to the perfusate of the alpha-adrenergic blocking agent, p h e n t o l a m i n e . Furthermore, phentolamine stimulated glucose induced insulin release to approximately the same extent in fed rats. Therefore, it is unlikely that catecholamines mediate the decreased insulin response to starvation. Our experiments also show that, in fasted rats, a 40 min equilibration with a high glucose concentration (1.5 mg/ml) induced marked enhancement of basal insulin release. However, even under such conditions the response was significantly lower than that of fed rats given 0.8 mg/ml of glucose during the preincubation. This finding suggests that the defective glucose induced insulin release on fasting cannot be ascribed to lack of substrate at the initiation of the stimulation. Acknowledgements. The excellent technical assistance of Mrs. Gila Gladnikoff, Mrs. Christina Hallgren and Mr. Aru Sandanam is acknowledged. Financial support was obtained from the Swedish Medical Research Council (grant no B 75-19X-34-11), the Nordic Insulin Foundation, and Knut and Alice Wallenberg's Foundation. 1. Coore , H.G. , Randle , P.J.: Regulation of insulin secretion studied with pieces of rabbit pancreas incubated in vitro . Biochem. J . 93 , 66 - 78 ( 1964 ) 2. Porte , D.: A receptor mechanism for the inhibition of insulin release by epinephrine in man . J. din. Invest . 46 , 86 - 94 ( 1967 ) 3. Loubatirres , A. , Mariani , M.M. , Chapal , J.: Etude physiologique et pharmacologique de l' insulino-srcr6tionsur le pancrras isol6 et perfus6 du rat. II. Effets des cat6cholamines et des substances bloquant les rrcepteurs adrrnergiques . J. Pharmacol. (Paris) 1 , 227 - 242 ( 1970 ) 4. Cerasi , E. , Luft , R. , Efendir , S. : Antagonism between glucose and epinephrine regarding insulin secretion . Acta med . scand. 190 , 411 -- 417 ( 1971 ) 5. Robertson , R.P. , Porte , D. : Adrenergic modulation of basal insulin secretion in man . Diabetes 22 , 1 - 8 ( 1973 ) 6. Christensen , N.J. , Iversen , J.: Release of large amounts of noradrenaline from the isolated perfused canine pancreas during glucose deprivation . Diabetologia 9 , 396 - 399 ( 1973 ) 7. Feldman , J.M. , Lebovitz , H. E. , Bowman , J.: Role of pancreatic monoamines in the impaired insulin secretion of the fasting state . Endocrinology 92 , 1469 - 1474 ( 1973 ) 8. Loubatirres , A. , Mariani , M.M. , Ribes , G., de Malbosc, H.: Etude exprrimentale d'un nouveau sulfamide hypoglycrmiant particulirrement actif , le HB 419 ou glibenclamide. I. Action brtacytotrope et insulinosrcrrtrice. Diabetologia 5 , 1 - 10 ( 1969 ) 9. Malaisse , W.J. , Malaisse-Lagae , F. , Wright , P.H. : Effect of fasting upon insulin secretion in the rat . Amer. J. Physiol . 213 , 843 - 848 ( 1967 ) 10. Grey , N.J. , Goldring , S. , Kipnis , D.M.: The effect of fasting, diet and actinomycin D on insulin secretion in the rat . J. clin. Invest . 49 , 881 - 889 ( 1970 ) 11. Voyles , N. , Gutman , R.A. , Selawry , H. , Fink , G. , Penhos , J. C. , Recant , L. : Interaction of various stimulators and inhibitors oh insulin secretion in vitro . Hormone Res . 4 , 65 - 73 ( 1973 ) 12. Bosbom , R.S. , Zweens , J. , Bouman , P.R.: Effects of feeding and fasting on the insulin secretory response to glucose and sulfonylureas in intact rats and isolated perfused rat pancreas . Diabetologia 9 , 243 - 250 ( 1973 ) 13. Hedeskov , C.J. , Capito , K. : The effect of starvation on insulin secretion and glucose metabolism in mouse pancreatic islets . Biochem. J . 140 , 423 - 433 ( 1973 ) 14. Fink , G. , Gutman , R.A. , Cresto , J.C. , Selawry , H. , Lavine , R. , Recant , L. : Glucose induced insulin release patterns: Effect of starvation . Diabetologia 10 , 421 - 425 ( 1974 )


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S. Efendić, E. Cerasi, R. Luft. Effect of phentolamine and preperfusion with glucose on insulin release from the isolated perfused pancreas from fasted and fed rats, Diabetologia, 1975, 407-410, DOI: 10.1007/BF00429908